sonic hedgehog

Question 1

How do secreting cells alter Shh before it is secreted?

Answer 1

N-terminal carbohydrate added in the golgi body followed by N-terminal palmitoylation by Skinny

Question 2

How is Shh released from cells? what restricts movement? How does it bind to the receiving cell?

Answer 2

Hh released from cells by Dispatched (multimer, heparan sulphate proteoglycan, permits EC transport)

Hh interacting protein (Hip) can restrict movement

iHog/Boi facilitate binding to receiving cell. (iHog/Boi mutants show defects in neural differentation and phenocopy Smo mutants > essential for Shh pathway)

Question 3

Described the receptor for Shh and explain what happens in resting state i.e. when No Shh is bound

Answer 3

Receptore is Patched.
12 span TM protein

In absence of Hh, Patched inhibits Smoothened (7 span TM protein)

Question 4

what happens when Shh binds to Patched receptor? (step one)

Answer 4

hh binding prevents Ptc inhibition of Smo, Smo is phosphorylated

Question 5

What happens after Smo is phosphorylated? (next step)

Answer 5

Smo activates Gli transcription factors

Question 6

Where is GliA usually?

Answer 6

Held in the cytoplasm by suppressor of fused (SuFu), also mediates slimb association and proteosome proccessing to repressor form GliR

Slimb = drosophila protein, targets Ci for ubiquitylation and protesome processing to repressor form CiR.

Question 7

Explain the negative feedback loop seen in Ptch/hh interaction

Answer 7

Ptc represses smo in the absence of Hh

Hh induces Gli activation

Ptc is a transcriptional target of Gli

More Ptc is inserted into the membrane, repressing Smo

Question 8

What is hh involved in in Drosophila?

Answer 8

Larval body segment development

Question 9

How does hh act in a paracrine fashion in drosophila?

Engrailed/Wingless/Ci

Answer 9

During body segment formation, stripes of cells synthesis Engrailed and also express Hh.

Hh cannot move far from where it is released so only activates a thins tripe of cells adjacent to Engrailed expressing cells

Only cells to one side of Engrailed-expressing cells are competent to respond to hh/Ptc interaction

Cells with activated Ptc receptors synthesise wingless

Wingless binds to Ci (Gli equivalent), results in incr wingless transcription of cells adj to stripe of Hh-prod cells

Wingless acts as an EC signal, patterns adjacent rows of cells by activating Frizzled (Wnt)
Wingless acts on engrailed-expressing cell to stabilise engrailed expression

Question 10

Why is hh signalling important in drosophila?

Answer 10

Reciprocal signalling of hh & wingless stabilises the boundary between parasegments.

Effects of wingless & Hh on other stripes of cells in each segment establishes positional code, accounts for anatomical features along the AP axis

Shh mutants= body like a hedhehog

Question 11

Why are cilia important for hedgehog signalling?

Answer 11

All the key components of hh signalling are enriched in cilia - Gli TFs, Smo & Ptc1

IFT is req. for Gli activation, esp. in limb development –> IFT mutants = polydactyly

BETH ADD MORE (LOOK AT DISSO)

Question 12

Where is Shh produced during neurulation? what does it cause?

Explain role of Shh in neural tube

Answer 12

Produced by the notochor, causes differentiation of ventral cells of Neural tube, important in creating gradients in neural tube formation

results in movement of axons towards ventral surface of developing spinal cord

Bending of NP occurs at MH & DLHP

MHP is induced by notocord signalling - sole site of bending in upper spinal neural plate

DLHP - SHH signalling from notocord inhibits DLHP formation at upper spinal level

As wave of spinal neurulation passes down the neuraxis, strength of Shh sinalling from notocord decreases, allowing DLHP formation to ‘break through’ at lower spinal levels

DL binding is negatively regulated by Shh - DLHP formation in lower spine can be inhib by local shh release

Supported by mice studies - Shh null mutant = DHLPs at every level and NT closes correctly

Shh overexpression can produce NTDs - inhibit DL binding in places essential for correct NT closure

Question 13

What does notocord Shh signalling induce in somitogenesis?

Answer 13

Induces ventral somite to differentiate into sclerotome

Ectopic expression found markers e.g. Pax1

Question 14

It is thought that Shh works with other Hh ligands, what do deleterious mutations in either/both ligands cause?

Answer 14

Wnt1,3,4 +Shh –> myogenesis

Shh KO = cyclopia/NTD/somite/foregut patterning defects

shh + Ihh work togerher, double KO = embryonically lethal –> heart morphogenesis malformation

Question 15

Which experiments formed the basis of the morphogen hypothesis?

Answer 15

Saunders & Gasseling (1969) on chick limb bud found that a diffusible factor was involved in development, later shown to be Hh

Question 16

Where is Shh produced during limb bud formation?

Answer 16

Shh is produced by ZPA (small region of tissue at posterior margin of the limb)

Question 17

Why is the ZPA significant during limb development?

Answer 17

Signalling within ZPA leads to development of the limb along AP axis

wnt7a is required for Shh in ZPA

Question 18

Why is the AER significant during limb development?

Answer 18

Part of ectoderm, secretes FGF8 which induces Shh and maintains the limb gradient dorsally

Question 19

How is shh involved in digit patterning?

Answer 19

Current model in murine embryos= concentration and time of Shh exposure determines which digits form

Works in an autocrine fashion (digits 5,4 and some of 3 arise from cells expressing shh)

Digit 5: longest expression, highest conc of Shh

Digit 4: Less time and conc of Shh (some diffused from 5)

Digit 3: Lesser so compared to 4&5

Digits 5,4&3 form in absence of Dispatched - autocrine signalling takes place, however Dispatched is required for EC diffusion to aid formation of other digits

Digit 2: receives EC Shh from digit 3 (has dispatched) (shh cant move far)

Digit 1: doesn’t require shh - default programme of limb bud cells

Question 20

How is hh signalling involved in eye development?

Answer 20

Shh involved in early stages of eye development

Eye develops from a population of cells from ANTERIOR NEURAL PLATE

Eye fields originally located centrally, separates into two fields located centrally.

Eye fields separate into two lateral optic vesicles

Shh secreted from the midline is required for separation of the eye field into 2 lateral optic vesicles

shh mutant mice = cyclopia
holoprosencephaly in humans

Question 21

How is hh signalling involved in ear development?

Answer 21

Shh antagonizes Wnt protein from roof plate which refines and maintains dorsoventral axial patterning in the ear.

Shh directly promotes the development of spiral ganglion neuron during neurogenesis. After Shh signaling pathway is activated, Shh indirectly upregulates Ngn1 expression > regulating neurogenic patterning of inner ear.

Shh regulates the differentiation of hair cells - influences the cell cycle of cochlea progenitor cells

Basal-to-apical wave of Shh decline ensures the normal development pattern of hair cells

Question 22

Experiments/ Evidence for shh involvement in NTDs

Answer 22

homozygous extra-toes mouse has a cranial neurulation defect. Mutant for Gli3 which negatively regulates shh signalling. Gli 1 & 2 mediate Shh > mutations do not lead to NTDs

Ptc-1 (shh off) mutation > ligand independent consitutive activation of shh signallling. Ptc-1 null mice = severe NTDs (same as Shh overexpression)

PKA = heteromeric enzyme, inhibits Shh signalling - decr PKA dose by 75% leads to a high freq of spinal defects >overrtim of shh inhibi DL binding of neural folds, leading to spina bifida and exencephaly

Question 23

There is an indication that Shh is neg reg DLHP formation at high spinal levels to ventralise closing NT & prevent dorsally located cells from differentiating in a direction necessary for DLHP formation … mutant studies?

Answer 23

Two other mutants: open-brain & zic2 = failure of NT closure in both brain & lower spine
Opb - disrupts rab23 gene (encodes neg reg of shh signalling)
Opb homozygous = dorsal & dorsolateral cell fates are not specified in developing NT, indicated by a lack of wnt3a & msx1 genes

Question 24

Shh regulation of neural fold bending is a mechanism operated at every level of the body axis

Answer 24

PKA = heteromeric enzyme, inhibits Shh signalling - decr PKA dose by 75% leads to a high freq of spinal defects >overrtim of shh inhibi DL binding of neural folds, leading to spina bifida and exencephaly

Question 25

How is shh involved in NS boundary formation?

Answer 25

ZLI = Zona limitans intrathalamica, at border of thalamus and prethalamus within the diencephalon

ZLI aries as a broad wedge of cells which narrows, marked by absence of Lfng expression (modulator of Notch signalling)

ZLI is a compartment - does not mix with adjacent thalamus

ZLI expresses Shh - Shh also expressed in floorplate for hindbrain and spinal cord forming neurons

shh induces region-specific gene expression in thalamus and prethalamus

Distinct gene expression due to different competence to respond to Shh

Question 26

How is Shh involved in cortical development?

Answer 26

Fate of neural stem/progenitor cells is determined by their position

Type of neuron and/or glial cell generated by the NSC/NPC is determined by the position of the cell in the developing brain, controlled by expression of BMP and Shh

Question 27

How is Shh involved in heart development?

Answer 27

shh & FGF are involved in compact layer formation in the compaction & trabeculation stage of heart development

Shh & Ihh work together - double KO is embryonically lethal due to heart morphogenesis malformation